核酸适配体识别-荧光法检测赭曲霉素A

建立了核酸适配体识别-荧光探针技术检测赭曲霉素A(OTA)的新方法.基于微孔板上固定的核酸适配子与目标物质OTA结合时构象发生变化,导致预先与其互补杂交的FAM标记短链DNA解离,引起荧光信号发生变化,据此可实现对OTA的定量检测.当微孔板包被亲和素浓度为25 mg/L、适配子浓度为50 nmol/L,FAM标记互补短...     

Aptamer-Based Fluorescence Nanoprobe for Sensitive and Selective Detection of Potassium

A new aptamer-based fluorescence nanoprobe for potassium ion (K⁺) has been developed. The nanoprobe employs gold nanoparticles (AuNPs) as the sensing platform and Rhodamine B as the fluorescence indicator. Aptamer acts as the switch of fluorescence signal of Rhodamine B. In the presence of K⁺, aptamer departs from AuNPs as a result of the formation of G-quartets with K⁺, leading to the decrease of fluorescence signals. Under the optimum conditions, the limit of detection (LOD) for K⁺ is as low as 3.8 nM. The proposed method was successfully applied in the determination of K⁺ in human saliva sample.     

基于核酸适配体的荧光法检测水胺硫磷和丙溴磷

建立了基于适配体的农药水胺硫磷和丙溴磷的荧光检测方法.采用可特异性识别水胺硫磷和丙溴磷、且5 '端标记荧光基团FAM的核酸适配体(F-ssDNA),与3 '末端标记猝灭基团DABCYL的短链序列(Q-ssDNA)互补杂交形成双链结构,荧光基团的荧光被淬灭,荧光信号很弱;此时加入靶分子,特异性结合核酸适配体,引起互补短链序列从双链结构中解离,使适配体荧光信号增强,基于此可实现水胺硫磷、丙溴磷的定量检测.优化后的检测条件为:将终浓度为25 nmol/L F-ssDNA与50 nmol/L Q-ssDNA在25℃孵育20 min,使二者杂交形成双链适配体探针复合物,加入等体积的农药样品孵育60 min,然后检测体系的荧光信号变化值△I.在最佳条件下,△I与水胺硫磷和丙溴磷的浓度均在50～ 500 μmol/L范围内呈线性关系.水胺硫磷的检出限(LOD,3σ)为11.4 μmol/L,相对标准偏差(RSD)为5.8％(n=10);丙溴磷的检出限为14.0 μmol/L,RSD为4.9％(n=l0).用于实际水样中两种农药的检测,加标回收率为85.8％ ～95.3％.     

Indirect Competitive Aptamer-Based Enzyme-Linked Immunosorbent Assay (apt-ELISA) for the Specific and Sensitive Detection of Isocarbophos Residues

Environmental contamination from pesticide residues is a worldwide public health concern and has attracted increasing attention from researchers over the past several years. In this study, an aptamer-based enzyme-linked immunosorbent assay (apt-ELISA) using enzyme signal amplification has been developed to determine isocarbophos residues. The limit of detection for the apt-ELISA was 0.05?ng/mL for isocarbophos, and the sensitivity was better than that obtained using the traditional ELISA approach. The average recovery of the apt-ELISA ranged from 84 to 97%, and the coefficient of variation ranged from 4.6 to 12.1% when determining the isocarbophos residues in various water samples. The results demonstrate that the proposed approach can be used for routine screening of isocarbophos residues.     

S,S-氰戊菊酯间接竞争酶联免疫吸附分析方法研究

合成了S,S-氰戊菊酯的半抗原S-2-(4-氯苯基)-3-甲基丁酸-α-S-(N-丁酸基)-甲酰氨-(3-苯氧基)苄酯(Efvb).半抗原通过混合酸酐法与卵清蛋白(OVA)偶联作为包被抗原,活泼酯法与牛血清蛋白(BSA)偶联作为免疫抗原.用免疫抗原免疫新西兰大白兔,得到抗S,S-氰戊菊酯多克隆抗体.通过异源分析及检测条件优化,确立了间接竞争酶联免疫分析方法的最佳检测条件为pH 7.4,0.4 mol/L Na+、40％甲醇-PBS溶液,建立了S,S-氰戊菊酯酶联免疫分析方法.方法的抑制中浓度(IC50)值为(3.16±0.01)mg/L;检出限(IC10)为(0.0053±0.0012) mg/L.对甲氰菊酯、溴氰菊酯、氯氰菊酯、三氟氯氰菊酯及其代谢物戊菊酸没有明显交叉反应.在自来水、河水和土壤样品中添加S,S-氰戊菊酯,其回收率分别为82.3％～108.2％,83.1％～109.2％和72.0％～91.2％.     

Fluorescence Assay of Proline: Competitive Inhibition of Fluorescamine Reaction

Abstract

The rate of reaction of fluorescamine with proline is faster than with primary amino acids. Proline competitively inhibits the fluorogenic reaction of primary amines with fluorescamine, the degree of inhibition being proportional to the amount of proline. Under the conditions described, 5×10^?7 M proline produces a 10% diminution of fluorescence. An assay of proline or hydroxyproline based on fluorescence inhibition would be more sensitive than colorimetric assays based on ninhydrin or fluorescamine.     

基于芘的荧光熄灭的单质碘荧光敏感膜的研究

单质碘熄灭固定于增塑的ＰＶＣ膜中的芘的荧光，且这种熄灭作用可逆。本据此研制了用于测定单质碘浓度的荧光敏感膜，最佳膜组成为２～４ｍｇ芘、５０ｍｇＰＶＣ粉、１００ｍｇ邻苯二甲酸二异辛酯，测定碘的浓度范围为２．２６×１０＾－５～１．０４×１０＾－３ｍｏｌ／Ｌ。此膜测定单质碘的重现性好，响应时间小于４０ｓ。除Ｆｅ＾３＋、Ｂｉ＾３＋外，其它常见离子均无干扰。将此膜用于食盐中碘的测定，结果令人满意。     

核酸适体识别荧光法检测汞离子

通过对已知的Ni2+适体的改造,发现了对Hg2+有较强结合活性的核酸适体N1,基于N1的二级结构进行改造,获得了具有较高活性的核酸适体N5,并建立了荧光检测方法。以荧光基团FAM标记核酸适体,在94℃变性5 min,室温(25℃)下复性30 min后,适配体与标记有荧光猝灭基团DABCYL的一段互补序列Q2结合(适体与Q2的浓度比为1∶3),加入系列浓度的Hg2+与互补序列竞争结合核酸适体,以荧光信号的变化定量分析Hg2+浓度。结果表明,N1与Hg2+结合呈线性,线性范围为1.25～20 mg/L;检出限为0.62 mg/L;以N1结构为基础改造合成的序列和结构均不同的适体N4,N5,N6,N7对Hg2+的识别结合活性均不同,其中适体N5与Hg2+结合活性最为灵敏,特异性高,线性范围为0.156～2.50 mg/L;检出限为78μg/L。     

Novel Immunochromatography Assay Based on Background Fluorescence Quenching for the Sensitive Determination of Serum Cystatin C

It is recognized that cystatin C is an effective marker for monitoring the glomerular filtration rate and the clinical diagnosis of various diseases. In this study, a novel immunochromatographic method has been established to achieve quantitative detection of serum cystatin C. Unlike conventional and traditional gold immune chromatographic assays (GICAs), a uniform layer of a fluorescent film was added to the solid phase, which has been designated as the background fluorescence quenching immune chromatographic assay (bFQICA). Under the optimized conditions, there was a good correlation between the fluorescence signal ratio (F1/F2) of the background fluorescence (F1) to the fluorescence signal at the detection line (F2) for cystatin C at concentrations from 0.0?ng/mL to 100?ng/mL with a correlation coefficient of 0.9977. The detection limit was 0.69?ng/mL, and the recovery values were 87.9–105%. The differences between the intra- and interbatch precision were less than 15% in three batches. In addition, after 120 serum samples were detected, there were no significant differences in the results obtained by bFQICA and immunoturbidimetry (t?=?0.963, p?=?0.338?>?0.05). This work demonstrates that bFQICA is a simple, sensitive, and accurate approach for the determination of serum cystatin C, providing a new approach for the clinical diagnosis of cystatin C.     

基于氧化石墨烯荧光适体传感器的胰岛素检测

采用荧光基团(FAM)标记的核酸适体作为识别元件,氧化石墨烯为淬灭剂,建立了一种高选择性、高灵敏度的核酸适体传感器.核酸适体与氧化石墨烯结合后,荧光淬灭,此时溶液无荧光;加入胰岛素后,溶液中荧光得到恢复.利用荧光分析法检测加入胰岛素前后,溶液中荧光强度的变化,获取了荧光适体传感器的线性度和灵敏度,实现对胰岛素浓度的测定.结果表明,在5×10-8 ～ 1×10-5 mol/L范围内,胰岛素的浓度与溶液中荧光强度有良好的线性关系,检出限为10 nmol/L.采用此方法检测胰岛素,操作简便,检测速度快,准确性高,选择性好,检出限低.     

Enzyme Assay by Fluorescence Quenching Release: A Novel Fluorometric Method

Abstract

When concentrated fluorescent dye is encapsulated in lecithin liposomes, the fluorescence is Largely self-quenched, The quenching is relieved when the liposomes are disrupted end the escapes. The fluorescence quenching release (FQR) is shown to be proportional to the amount of phospholipase C of C. welchii which hydrolyses lecithin. The FQR method is more sensitive, rapid, and convenient than conventional titrimetric assay and is amenable to automation and Kinetic studies. As a general method, FQR could be adapted to the measurement of other enzymes or agents which disrupt dye-containing microstructures.     

荧光共振能量转移猝灭法测定加替沙星

在pH=7.00的Britton-Robinson (B-R)缓冲溶液,及十二烷基硫酸钠(SDS)介质中,吖啶橙(AO)-罗丹明 (RB)能够发生有效的能量转移,使RB荧光增强.加替沙星(Gatifloxacin, GTFX)的加入又使得RB的荧光猝灭,据此建立了测定加替沙星的新方法.将此方法用于加替沙星片剂和粉针剂的测定,结果满意.实验表明,该方法简单、快速、灵敏、准确.     

荧光-噻唑蓝法检测红色纳米硒的抗氧化活性

采用荧光法检测红色纳米硒的抗氧化性,并结合噻唑蓝法(MTT法)研究红色纳米硒抗氧化对细胞的作用。Fenton反应产生稳定羟自由基,罗丹明B为荧光指示剂,磷酸盐缓冲液中检测了纳米硒清除羟自由基的效果。再将纳米硒与肝细胞QSG7701共培养,检测了不同时期培养基的氧化还原状况,并用MTT实验检测作用后细胞生长情况。结果显示:纳米硒具有良好抗氧化作用,能有效改善培养基中的氧化还原环境,同时促进了细胞生长。     

荧光共振能量转移猝灭法测定片剂中莫西沙星含量

基于在pH 6.50的B-R缓冲溶液,十二烷基硫酸钠(SDS)介质中,吖啶橙(AO)-罗丹明B(RB)能够发生有效的能量转移,使RB荧光增强,而加入莫西沙星(MXFC)后使得RB的荧光猝灭,且其荧光淬灭程度与莫西沙星的质量浓度在1.00～10.0mg.L-1范围内呈线性关系,据此提出了利用AO-RB荧光共振能量转移,荧光淬灭法间接测定片剂中莫西沙星含量的方法。方法的检出限(3S/N)为0.039mg.L-1。方法用于莫西沙星片剂的分析,测定值与高效液相色谱法测定值相一致,加标平均回收率为99%,相对标准偏差(n=5)均小于0.9%。     

Label-Free and Simple G-quadruplex-based Turn-Off Fluorescence Assay for the Detection of Kanamycin

We have developed a label-free and turn-off fluorescence assay for the determination of kanamycin. The detection system consists of an aptamer for specifically recognizing kanamycin and two auxiliary probes functionalized with two GGG repeats at the 3′ or 5′ ends for signal reporting. Two probes both hybridize with the aptamer and then their G-rich sequences combine to form a G-quadruplex. When thioflavin T, a fluorophore, is bound to the G-quadruplex, the fluorescence intensity of the solution dramatically increases. Upon the addition of the kanamycin, the aptamer–kanamycin binding inhibits the hybridization of two probes and aptamer, and restrains the GGG repeats from getting closer to form the G-quadruplex structure, resulting a significant decrease in the fluorescence intensity. The proposed aptamer-based fluorescent sensing platform showed a linear relationship with the concentration of kanamycin from 0.6 to 20.0?nM. The detection limit was determined to be 0.33?nM. The sensing platform provides resistance to interferences from other antibiotics and can be used to efficiently recognize kanamycin in real samples.     

Quenching of Pyrene Fluorescence in Premiceljar Solutions

Abstract

The quenching of the fluorescence intensity of pyrene by KBr has been measured in premicellar solutions of cetyltrimethylammonium bromide (CTAB), sodium lauryl sulfate (SLS) and polyoxyethylene 23-lauryl ether (Brij 35). The association of pyrene with premicellar aggregates is thought to bring the bromide ion closer to the fluorophore and hence results in a greater quenching effect. In 1.0 × 10^?4 M CTAB solutions there is the beginning of “protective” premicellar aggregation. At this stage, 13 times more bromide ion is needed to effect the same degree of quenching as in pure water.     

Aptamer-mediated turn-on fluorescence assay for prion protein based on guanine quenched fluophor

An aptamer-participated haprin structure was designed by employing cellular prion protein (PrP^C) as a model protein, and thus an aptamer-mediated turn-on fluorescence assay for proteins was developed in this contribution. The designed aptamer-participated haprin structure consists of three segments. Namely, an aptamer sequence located in the loop, three guanine bases at 3′-terminal, and a fluophor modified at 5′-terminal. It was found that the guanine bases at the 3′-terminal could quench the fluorescence of the fluophor such as tetramethyl-6-carboxyrhodamine (TAMRA) at the 5′-terminal about 76.6% via electron transfer if the guanine bases are close enough to the fluophor, and the quenched fluorescence could get restored when the target protein is present since the interaction, which could be confirmed by measuring fluorescence lifetime, between TAMRA-aptamer and the target protein forces the guanines away from TAMRA so that TAMRA-modified aptamer changes into turn-on state. A linear relationship was then constructed between the turn-on fluorescence intensity and the concentration of PrP^C in the range from 1.1 to 44.7 μg/mL with a limit of detection of 0.3 μg/mL (3σ).     

Determination of Sodium Hexametaphosphate in Beverages Based on the Fluorescence Quenching of Acridine Orange

Sodium hexametaphosphate was shown to form a complex with acridine orange by electrostatic interactions that induce fluorescence quenching. Analysis of fluorescence intensity showed that the process was dominated by static quenching, which was confirmed by absorption spectra and lifetime of the excited state. The decreased fluorescence intensity was directly proportional to the concentration of sodium hexametaphosphate between 8.0 × 10^?7 and 1.1 × 10^?5 mol L^?1 with a limit of detection of 5.3 × 10^?7 mol L^?1. Beverages were analyzed for sodium hexametaphosphate with recoveries between 91.7% and 108.3%.     

Fluorescence Assay for Membrane Lytic Peptides

Abstract

Lytic peptides such as melittin and mastoparan are usually assayed by measuring the leakage of cell contents; e.g., hemolysis. When such peptides lyse liposomes containing concentration-quenched 6-carboxyfluorescein (6CF), the resulting fluorescence increase is proportional to the amount of lytic peptide added. Using this 6CF-liposome system, one can assay nanogram quantities of melittin. A protocol was developed to survey peptides for lytic activity and at the same time, to test for mast cell degranulating activity. Peptides possessed either, both, or neither of these activities. The dye-liposome system was used to assay HPLC fractions of bee venom. This fluorescence assay for lytic activity is more sensitive and convenient than the hemolysis method, does not require removal of unlysed structures, and does not require animal cells.     

Time-Resolved Fluorescence Receptor Assay for Benzodiazepines

Abstract

A novel non-isotopic receptor binding technique for the detection of benzodiazepines is described. A benzodiazepine labeled with europium chelate was prepared and employed as a labeled ligand, and time-resolved measurements of the long lifetime fluorescence of europium chelate allowed avoidance of interference due to proteins in the receptor preparation. Experimental results demonstrate a sigmoid inhibition curve, with binding of the labeled ligand inhibited by comparable concentrations of the unlabeled drug. The proposed assay may provide a simple procedure for the measurement of benzodiazepines in biological systems and a useful tool in the screening of natural substances for new classes of benzodiazepine-like compounds.